Human and mouse embryonal carcinoma cells and early embryo cells display a unique phenotype: they do not express certain viral and cellular genes that are expressed in differentiated successors. Therefore, they provide a system to study interesting aspects of the control of gene expression during very early embryonic development. We have found evidence that primary control of gene expression in the EC cell occurs by mechanisms other than de novo methylation. Although de novo methylation occurs, it may be a consequence of nonexpression. We will study the mechanisms of this transcription block in EC cells by introducing various constructs of cloned genes into EC cells by protoplast fusion to overcome the restriction. The results should distinguish between cis and trans acting control mechanisms in the cell. Cytosine residues in DNA will be replaced with 5-azacytidine by growing hybrid plasmids in media containing the analog. The expression of these genes will be tested in EC cells and compared to expression of the sequences without the nucleotide analog. A cloned "housekeeping" gene, CAD, will be transformed into EC cells to determine whether it is expressed. The CAD gene presumably is expressed already by EC cells and the procedure should tell if all exogenous genes are selectively not expressed by EC cells. Proviral sequences will be introduced into a hybrid plasmid vector that has been constructed to express neomycin resistance in EC cells. We may find that expression of the proviral genes is accomplished only under the influence of other active promotors. Promotor enhancer sequences from polyoma virus mutants that grow in EC cells will be linked covalently to provirus and their effect on gene expression in EC cells will be examined. If results indicate that the enhancer sequences will promote transcription of proviral genes, they will be inserted into the provirus to construct a retroviral vector that will express exogenous genes in EC and early embryonic stem cells.